Blood vessels are the means by which oxygen and nutrients are supplied to living tissues and waste products are removed from living tissue. Angiogenesis refers to the process by which new blood vessels are formed. See, for example, the review by Folkman and Shing, J. Biol. Chem. 267, 10931-10934 (1992). Thus, where appropriate, angiogenesis is a critical biological process. It is essential in reproduction, development and wound repair. However, inappropriate angiogenesis can have severe negative consequences. For example, it is only after many solid tumors are vascularized as a result of angiogenesis that the tumors have a sufficient supply of oxygen and nutrients that permit it to grow rapidly and metastasize. Because maintaining the rate of angiogenesis in its proper equilibrium is so critical to a range of functions, it must be carefully regulated in order to maintain health. The angiogenesis process is believed to begin with the degradation of the basement membrane by proteolytic enzymes, e.g., metalloproteinases (MMPs) and plasminogen activator (PA), secreted from endothelial cells (EC) activated by mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
Proteolytic activity is also required for the migration of EC into the perivascular stroma. These events are followed by sprout extension and subsequent lumen formation (Ausprunk, D. H., et al., Microvascular Res. 14:53-65 (1977)). As is EC “escape” from the parent venule, capillary sprout elongation, lumen formation, and EC migration are all events which are dependent on a shift in the proteolytic balance in favor of enzymatic activity (Ausprunk, D. H., et al., Microvascular Res. 14:53-65 (1977), Kalebic, T., et al. Science, 221:281-283 (1983), and (Moses, M. A., et al., Science 248:1408-1410 (1990)). Vascular morphogenesis and invasion are also regulated by shifts in the finely tuned balance between proteases and their inhibitors (Liotta, L. A., et al., Cell 64:327-336, (1991); Moses, M. A., et al., J. Cell Biochem. 47:1-8 (1991); Herron, G. S., et al., J. Biol. Chem. 261:2814-2828 (1986), and Montesano, R., et. al., Cell 62:435-445, (1990)).
An accumulating body of evidence suggests that the remodeling of ECM that occurs during normal growth, wound repair and angiogenesis as well as during the development and progression of pathologic conditions including malignant diseases, is accomplished largely through the action of MMPs (Birkedal-Hansen, H. Cell. Bio. 7:728-735 (1985), Matrisian, L. Trends Genet. 6:121-125, (1990), and Woessner, J. F. Acad. Sci. 732:11-21 (1994), and Woessner, J. F. Ann. N.Y. Acad. Sci. 732:11-21, 1994).
The MMPs are members of a multigene family of metal-dependent enzymes. These proteases have been classified into four broad categories originally based on substrate specificity. These specific enzymes are the collagenases (MMP-1/EC3.4.24.7; MMP-8/EC3.4.24.34; MMP-13), the gelatinases A (MMP-2/EC3.4.24.24) and B(MMP-9/EC3.4.24.35), the stromelysins (MMP-3/EC3.4.24.17:MMP-10/EC3.4.24.22; MMP-1/EC3.4.24.7) including a metalloelastase (MMP-12), the membrane MMPs (MMP-14) (Birkedai-Hansen, H. Current Opinions in Cell Biol. 7:728-735, 1995. Matrisian, L. Trends Genet. 6:121-125, 1990. Woessner, J. F. Ann. N.Y. Acad. Sci. 732:11-21, 1994) and the family of membrane type MMPs (MT-MMP 1-6).
The regulation of MMP activity occurs at several levels including gene transcriptional control, proenzyme activation and inhibition of activated MMPs by endogenous inhibitors. Like many other enzyme families, the MMPs are a key component of a system of “balanced proteolysis” wherein a finely tuned equilibrium exists between the amount of active enzyme and its proteinase inhibitor(s) (Liotta, L. A., et al., Cell 64:327-336, (1991)). These native metalloproteinase inhibitors comprise a family of proteins generally referred to as the TIMPS (Tissue Inhibitor of MetalloProteinase) (Docherty, A. J. P., et al., Nature 318:66-69, (1985), Carmichael, D. F., et al. Proc. Natl. Acad. Sci. USA 83:2407-2411, (1986); Moses, M. A., et al., J. Cell. Biochem. 47:230-235, (1991); Murray, J. B., et al., J. Biol. Chem. 261:4154-4159 (1986); Stetler-Stevenson, W. G., et al., J. Biol. Chem. 29:17374-17378, (1989); Pavloff, N., et al., J. Biol. Chem. 267:17321-17326, (1992), and DeClerck, T. A., et al., J. Biol. Chem. 264:17445-17453 (1989)). They bind to activate MMPs with 1:1 molar stoichiometry.
The TIMPs consist of six disulfide-bonded loops. Deletion mutagenesis studies have demonstrated that two structurally distinct domains can be defined, the N-terminal domain consisting of loops 1-3 and the C-terminal domain consisting of loops 4-6 (Murphy, G. Houbrechts., et al. Biochemistry 30(33):8097-8101, (1991); Willenbrock, F., et al., Biochem. 32:4330-4337, (1993), and Nguyen, Q., et al., Biochem. 33:2089-2095, (1994)).
Much research attention has been focused on studies aimed at defining the domains of TIMPs that are important to their ability to inhibit MMP activity. Construction of truncated forms of these molecules has provided some insight. Residues 1-126 of TIMP-1 and 1-127 of TIMP-2 which contain three of the six disulfide bonds in the full-length molecules have been expressed in mammalian cells in the absence of the G-terminal region and are secreted in a soluble form (Murphy, G., et al. Biochemistry 30(33):8097-8101, (1991)). These truncated forms inhibit matrilysin and the catalytic domains of stromelysin and gelatinase A, demonstrating that there is a direct interaction between the N-terminal domain of the TIMPs and the catalytic domains of the MMPs (Murphy, G., et al. Biochemistry 30(33):8097-8101, (1991); Willenbrock, F., et al., Biochem. 32:4330-4337, (1993), and Nguyen, Q., et al., Biochem. 33:2089-2095, (1994). The structure of the N-terminal domain of either TIMP-1 or TIMP-2 is not affected by the C-terminal domain (Nguyen. Q., et al., Biochemistry 33:2089-2095, (1994)).
A significant number of mutational analyses also support the concept that the NH2-terminal domain of TIMP-1 (Cys1-Glu126) is sufficient for inhibition of MMPs (Wilhelm, S. M., et al., J. Biol. Chem. 264:17213-17221, (1989), Murphy, G., et al., Biochemistry 30(33):8097-8101, (1991); Murphy, G., et al., Bio. Chem. Biophys. Acta. 839:214-218, (1985); Stricklin, G. P., Collagen Relat. Res. 6:219-228, (1986); Tolley, S. P., et al., Protein: Struc., Fuct., Genet. 17:435-437, (1993), and Howard, E. W., et al., J. Biol. Chem. 266:13064-13069, (1991)). Furthermore, single-residue mutations in the region bounded by Cys3 and Cys13 caused an increase of 8-fold in Ki when compared with wild type TIMP-1 (O'Shea, M., et al., Biochemistry 31(42):10146-10151, (1992)). A series of experiments including competition studies with synthetic peptides and localization of epitopes of blocking antibodies revealed that the region marking the transition between the NH2-terminal and COOH-terminal domains of the TIMP-1 molecule may be particularly important for its ability to inhibit collagenase (Bodden, M. K., et al., J. Biol. Chem. 269:18943-18952, (1994)).
It is now widely accepted that the N-terminal domain of the TIMPs represent a stable, minimized form of the inhibitor that includes the major site or sites necessary for MMP inhibition (Williamson, R. A., et al., Biochem. 33:11745-11759, (1994)). Site-directed mutagenesis studies on TIMP-1 have demonstrated that no single residue is likely to be responsible for MMP inhibition (O'Shea, M., et al., Biochem. 31(42): 10146-10151, (1992)).
The C-terminal domain of TIMPs also makes some binding contribution to the TIMP-MMP complex, in particular, the C-terminal domain of TIMP-2 which may be responsible for the specific interaction of this molecule with progelatinase A (Willenbrock, F., et al., Biochemistry 32:4330-4337, (1993)). Mutational studies also support the idea that the COOH-terminal domain of TIMP-2 which does not appear to be required for MMP inhibition (O'Shea, M., et al., Biochemistry 31(42):10146-10151 (1992)) interacts with the pexin-like domain of gelatinase A (Hayakawa, T., et al., FEBS Lett. 298:29-32, (1992)). This interaction has been shown to prevent autodegradation of the enzyme (Goldberg, G. I., et al., J. Biol. Chem. 267:4583-4591 (1992), Bodden, M. K., et al., Biol. Chem. 269:18943-18952, (1994), and Howard, E. W., et al., J. Biol. Chem. 266:13084-13089, (1991)).
TIMPs have been shown to inhibit the migration of endothelial cells in vitro and, depending on the model used, angiogenesis in vivo. Mignatti, et al. (J. Cell Bio. 108:671-682, (1989)) first demonstrated that TIMP-1 could inhibit migration of microvascular cells in vitro using the amnion invasion assay. Montesano and coworkers later showed that a collagenase inhibitor (1,10-phenanthroline) could inhibit capillary tube formation in vitro (Montesano, R., et. al. Cell 42:489-477, (1985)). Since it was first demonstrated that a TIMP purified from a vascular cartilage was a potent inhibitor of angiogenesis in vivo and EC proliferation and migration in vitro (Moses, M. A., et al., Science 248:1408-1410, (1990); Moses, M. A., et al., J. Cell Biol. 119:475-482, (1992) and Murphy, A. N. et al., J. Cell. Phys. 157:351-358 (1993)) showed that TIMP-2, but not TIMP-1, inhibited FGF-stimulated endothelial cell proliferation. TIMP-1 has been shown to stimulate the growth of EC proliferation in other studies as well (Hayakawa, T., et al FEBS Letts. 298:29-32, (1992)). TIMPs have been shown to inhibit neovascularization in various in vivo models (Takigawa, M., et al., Biochem. Biophys. Res. Commun. 171:1264-1271 and Johnson, M. D. et al., J. Cell. Physiol. 160:194-202, (1989)).
Much research attention has focused on studies aimed at defining the domain of TIMPs that are important to their ability to inhibit MMP activity. It is now widely accepted that the N-terminal domain of the TIMPs represents a stable, minimized form of the inhibitor that includes the major site or sites necessary for MMP inhibition (Williamson, R. A. et al. Biochemistry 33:11745-11759 (1994)). The C-terminal domain of TIMPs also makes some binding contribution to the TIMP-MMP complex, in particular, the C-terminal domain of TIMP-2 which may be responsible for the specific interaction of this molecule with progelatinase A (Willenbrock, F. et al., Biochemistry 32:4330-4337 (1993)).
It has been suggested that the regions of amino acid sequences between TIMP-1 and TIMP-2 that are highly conserved such as the N-terminus, may be responsible for the known shared functions of these proteins, for example, inhibition of activated MMPs and their shared ability to inhibit FGF-stimulated capillary EC migration (Moses, M. A. et al., Science 248:1408-1410 (1990); Moses, M. A. et al., J. Cell Biol. 119:475-482 (1992); Mignatti, P. et al., J. Cell Bio. 108:671-682 (1989) and Murphy, A. N. et al., J. Cell. Phys. 157:351-358 (1993)). Areas of low homology, for example, the C-terminus, may be responsible for those functions which are unique for the individual TIMPs (Stetler-Stevenson et al., J. Biol. Chem. 265(23)13933-13936 (1990)). These include the binding of TIMP-2 to the latent form of gelatinase A and the failure of TIMP-2 antibodies to detect TIMP-1 (Stetler-Stevenson, W. G. et al., J. Biol. Chem. 265(23):13933-13936 (1990).
The rate of angiogenesis involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels. The therapeutic implications of angiogenic growth factors were first described by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 285:1182-1186 (1971)). Abnormal angiogenesis occurs when the body loses at least some control of angiogenesis, resulting in either excessive or insufficient blood vessel growth. For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing. In contrast, excessive blood vessel proliferation can result in tumor growth, tumor spread, blindness, psoriasis and rheumatoid arthritis.
Thus, there are instances where a greater degree of angiogenesis is desirable-increasing blood circulation, wound healing, and ulcer healing. For example, recent investigations have established the feasibility of using recombinant angiogenic growth factors, such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF) (Pu, et al., J Surg Res, 54:575-83 (1993)), and more recently, vascular endothelial growth factor (VEGF) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70 (1994)).
Conversely, there are instances, where inhibition of angiogenesis is desirable. For example, many diseases are driven by persistent unregulated angiogenesis, also sometimes referred to as “neovascularization.” In arthritis, new capillary blood vessels invade the joint and destroy cartilage. In diabetes, new capillaries invade the vitreous, bleed, and cause blindness. Ocular neovascularization is the most common cause of blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.
The current treatment of these diseases is inadequate. Agents which prevent continued angiogenesis, e.g., drugs (TNP-470), monoclonal antibodies, antisense nucleic acids and proteins (angiostatin and endostatin) are currently being tested. See, Battegay, J. Mol. Med., 73, 333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman, N. Engl. J. Med., 333, 1757-1763 (1995). Although preliminary results with the antiangiogenic proteins are promising, they are relatively large in size and they are difficult to use and produce. Moreover, proteins are subject to enzymatic degradation. Thus, new agents that inhibit angiogenesis are needed. New antiangeogenic peptides that show improvement in size, ease of production, stability and/or potency would be desirable.